Abstract 343: Induction of Apoptosis in Human Macrophages by 7-ketocholesterol and Iron Ascorbate
Inflammation is the new paradigm for the development and progression of atherosclerotic lesions in coronary artery disease. Factors that increase oxidative stress and apoptosis of cells within the lesion contribute to sustaining the inflammatory response. It was hypothesized that redox-active iron and oxysterols, which have both been found in association with atherosclerotic plaques, contribute to oxidative stress and apoptosis by distinct, but overlapping signaling pathways. The human acute monocytic leukemia cell line THP-1 was used as a model system to study the roles of redox-active iron and 7-ketocholesterol in macrophage apoptosis. The ability of both reagents to induce apoptosis in THP-1 macrophages was demonstrated using flow cytometry. The kinetics of poly(ADP-ribose) polymerase (PARP) cleavage as measured by ELISA were also similar. qRT-PCR was used to measure mRNA levels of several pro- and anti-apoptotic genes. While 7-ketocholesterol increased the level of CHOP mRNA (a transcription factor in endoplasmic reticulum (ER)-initiated apoptosis), there was no similar increase in response to iron ascorbate treatment. Expression of other markers of ER stress, like the transcription factors ATF3 and CEBPbeta, was increased by both compounds, but the timing of changes in gene expression was different. TNFalpha regulates both apoptosis and inflammatory cytokine production. Macrophages are major producers of TNFalpha as well as being highly responsive to the cytokine. A comparison of the changes in TNFalpha mRNA and protein levels in response to iron ascorbate and 7-ketocholesterol suggested that while ERK1/2 and NFkB signaling are important in TNFalpha expression, differentially regulated post-transcriptional processes determine the release of TNFalpha by macrophages. These preliminary results suggest that the apoptotic pathways activated by oxysterols and redox-active iron may be different and that these compounds may have additive or synergistic effects on lesion progression.
- © 2012 by American Heart Association, Inc.